The present invention relates in general to surgical instruments, surgical techniques, and cell and tissue isolation techniques. More particularly, the present invention is directed to a surgical tool for transplanting retinal cells, epithelium and choroidea within their normal planar configuration, a graft for transplantation to the subretinal region of the eye, a method for preparing such grafts for transplantation, and a method for reconstructing dystrophic retinas, retinal pigment epithelial layers and choroids.
The retina is the sensory epithelial surface that lines the posterior aspect of the eye, receives the image formed by the lens, transduces this image into neural impulses and conveys this information to the brain by the optic nerve. The retina comprises a number of layers, namely, the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor inner segments and outer segments. The outer nuclear layer comprises the cell bodies of the photoreceptor cells with the inner and outer segments being extensions of the cell bodies.
The choroid is a vascular membrane containing large branched pigment cells that lies between the retina and the sclerotic coat of the vertebrate eye. Immediately between the choroid and the retina is the retinal pigment epithelium which forms an intimate structural and functional relationship with the photoreceptor cells.
Several forms of blindness are primarily related to the loss of photoreceptor cells caused by defects in the retina, retinal pigment epithelium, choroid or possibly other factors (e.g. intense light, retinal detachment, intraocular bleeding). In several retinal degenerative diseases select populations of cells are lost. Specifically, in macular degeneration and retinitis pigmentosa retinal photoreceptors degenerate while other cells in the retina as well as the retina""s central connections are maintained. In an effort to recover what was previously thought to be an irreparably injured retina, researchers have suggested various forms of grafts and transplantation techniques, none of which constitute an effective manner for reconstructing a dystrophic retina.
The transplantation of retinal cells to the eye can be traced to a report by Royo et al., Growth 23: 313-336 (1959) in which embryonic retina was transplanted to the anterior chamber of the maternal eye. A variety of cells were reported to survive, including photoreceptors. Subsequently del Cerro was able to repeat and extend these experiments (del Cerro et al., Invest. Ophthalmol. Vis. Sci. 26: 1182-1185, 1985). Soon afterward Turner, et al. Dev. Brain Res. 26:91-104 (1986) showed that neonatal retinal tissue could be transplanted into retinal wounds.
In related studies, Simmons et al., Soc. Neurosci. Abstr. 10: 668 (1984) demonstrated that embryonic retina could be transplanted intracranially, survive, show considerable normal development, be able to innervate central structures, and activate these structures in a light-dependent fashion. Furthermore, these intracranial transplants could elicit light-dependent behavioral responses (pupillary reflex) that were mediated through the host""s nervous system. Klassen et al., Exp. Neurol. 102: 102-108 (1988) and Klassen et al. Proc. Natl. Acad., Sci. USA 84:6958-6960 (1987).
Li and Turner, Exp. Eye Res. 47:911 (1988) have proposed the transplantation of retinal pigment epithelium (RPE) into the subretinal space as a therapeutic approach in the RCS dystrophic rat to replace defective mutant RPE cells with their healthy wild-type counterparts. According to their approach, RPE were isolated from 6- to 8-day old black eyed rats and grafted into the subretinal space by using a lesion paradigm which penetrates through the sclera and choroid. A 1 xcexcl injection of RPE (40,000-60,000 cells) was made at the incision site into the subretinal space by means of a 10 xcexcl syringe to which was attached a 30 gauge needle. However, this method destroys the cellular polarity and native organization of the donor retinal pigment epithelium which is desirable for transplants.
del Cerro, (del Cerro et al., Invest. Ophthalmol. Vis. Sci. 26: 1182-1185, 1985) reported a method for the transplantation of tissue strips into the anterior chamber or into the host retina. The strips were prepared by excising the neural retina from the donor eye. The retina was then cut into suitable tissue strips which were then injected into the appropriate location by means of a 30 gauge needle or micropipette with the width of the strip limited to the inner diameter of the needle (250 micrometers) and the length of the strip being less than 1 millimeter. While del Cerro reports that the intraocular transplantation of retinal strips can survive, he notes that the procedure has some definite limitations. For instance, his techniques do not allow for the replacement of just the missing cells (e.g. photoreceptors) but always include a mixture of retinal cells. Thus, with such a transplant appropriate reconstruction of the dystrophic retina that lacks a specific population of cells (e.g., photoreceptors) is not possible.
del Cerro et al., Neurosci. Lett. 92: 21-26, 1988, also reported a procedure for the transplantation of dissociated neuroretinal cells. In this procedure, the donor retina is cut into small pieces, incubated in trypsin for 15 minutes, and triturated into a single cell suspension by aspirating it through a fine pulled pipette. Comparable to the Li and Turner approach discussed above, this procedure destroys the organized native structure of the transplant, including the donor outer nuclear layer; the strict organization of the photoreceptors with the outer segments directed toward the pigment epithelium and the synaptic terminals facing the outer plexiform layer are lost. Furthermore, no means of isolating and purifying any given population of retinal cells (e.g. photoreceptors) from other retinal cells was demonstrated.
It is believed by the present inventor that it is necessary to maintain the photoreceptors in an organized outer nuclear layer structure in order to restore a reasonable degree of vision. This conclusion is based on the well known optical characteristics of photoreceptors, (outer segments act as light guides) and clinical evidence showing that folds or similar, even minor disruptions in the retinal geometry can severely degrade visual acuity.
Among the objects of the present invention, therefore, may be noted the provision of a method for preparation of a graft for use in the reconstruction of a dystrophic retina; the provision of such a method which conserves relatively large expanses of the tissue harvested from a donor eye; the provision of such a method in which the polarity and organization of the cells at the time of harvest are maintained in the graft; the provision of a graft for use in the reconstruction of a dystrophic retina; the provision of such a graft which facilitates regrowth of photoreceptor axons by maintaining the polar organization of the photoreceptor and the close proximity of their postsynaptic targets with the adjacent outer plexiform layer upon transplantation; the provision of a surgical tool for use in the transplantation method which allows appropriate retinotopic positioning and which protects photoreceptors or other grafted tissue from damage prior to and as the surgical device is positioned in the eye; and the provision of a method for transplantation of grafts to the subretinal area of an eye.
Briefly, therefore, the present invention is directed to a method for the preparation of a graft for transplantation to the subretinal area of a host eye. The method comprises providing donor tissue and harvesting from that tissue a population of cells selected from retinal cells, epithelial tissue or choroidal tissue, the population of cells having the same organization and cellular polarity as is present in normal tissue of that type. The population of cells is laminated to a non-toxic and flexible composition which substantially dissolves at body temperature.
The present invention is further directed to a method for the preparation of a graft comprising photoreceptor call bodies for transplantation to the subretinal area of a host eye. The method comprises providing a donor retina containing a layer of photoreceptor cell bodies. The layer of photoreceptor cell bodies is isolated from at least one other layer of cells of the donor retina in a manner that maintains the layer of photoreceptor cell bodies in the same organization and cellular polarity as is present in normal tissue of that type.
The present invention is further directed to a graft for transplantation to the subretinal area of a host eye. The graft comprises a laminate of a non-toxic and flexible composition which substantially dissolves at body temperature and a population of cells harvested from a donor eye, the population of cells being selected from retinal cells, epithelial tissue and choroidal tissue. The population of cells has the same organization and cellular polarity as is present in normal tissue of that type.
The present invention is further directed to a graft for transplantation to the subretinal area of a host eye. The graft comprises a population of photoreceptor cell bodies harvested from a donor retina, the population of photoreceptor cell bodies having the same organization and cellular polarity as is present in normal tissue of that type, the graft having an essential absence of at least one layer of cells present in the donor retina.
The present invention is further directed to a method for transplanting to the subretinal area of a host""s eye a graft comprising a population of cells. The method comprises providing a graft comprising a population of cells selected from retinal cells isolated from at least one other layer of cells within the retina, epithelial tissue and choroidal tissue, the population of cells being maintained in the same organization and cellular polarity as is present in normal tissue of that type. An incision is made through the host""s eye, the retina is at least partially detached to permit access to the subretinal area and the graft is positioned in the accessed subretinal area.
The present invention is further directed to an instrument for the implantation of an intact planar cellular structure between the retina and supporting tissues in an eye. The instrument comprises an elongate supporting platform for holding the planar cellular structure. The platform has a distal end for insertion into an eye, and a proximal end. The distal edge of the platform is convexly curved for facilitating the insertion of the platform into the eye and the advancement of the platform between the retina and the supporting tissues. The instrument has a side rail on each side of the platform for retaining the planar cellular structure on the platform, the distal ends of the side rails being rounded, and the distal portions of the side rails tapering toward the distal end of the platform to facilitate the insertion of the instrument between the retina and the supporting tissue.
The present invention is further directed to an instrument for the implantation of an intact planar cellular structure between the retina and supporting tissues in an eye. The instrument comprises an elongate tube, having a flat, wide cross-section, with a top, a bottom for supporting the planar cellular structure, and opposing sides. The tube has a beveled distal edge facilitating the insertion of the tube into the eye and the advancement of the tube between the retina and the supporting tissues. The instrument also comprises plunger means for ejecting a planar cellular structure from the distal end of the tube.
The present invention is further directed to a kit for transplantation of a graft to the subretinal area of a host eye. The kit contains a graft comprising a population of cells selected from retinal cells, epithelial tissue and choroidal tissue, the population of cells being maintained in the same organization and cellular polarity as is present in normal tissue of that type. The kit additionally contains a surgical instrument comprising an elongate tube, having a flat, wide cross-section, with a top, a bottom for supporting the planar cellular structure, and opposing sides. The tube has a beveled distal edge facilitating the insertion of the tube into the eye and the advancement of the tube between the retina and the supporting tissues. The surgical instrument also comprises plunger means for ejecting a planar cellular structure from the distal end of the tube.
Other objects and features of the invention will be in part apparent and in part pointed out hereinafter.